Dysfunction of the lysosomal system (LS) in inherited human disease is usually associated with neurodegeneration and cognitive decline. In Alzheimer's disease, we have identified distinctive LS abnormalities that develop early in neurons at risk to degenerate, progress with the disease, and are accentuated in types of familial AD. The overall objectives of this project are to test the hypothesis that neuronal LS dysfunction in AD leads to cell atrophy and promotes modified mice, and neuronal cell culture models. Using specific immunological probes of LS function and confocal microscopy applied to well-characterized human brains in the earliest stages of AD, we will establish the temporal and spatial relationship between LS changes, and Abeta, beta-amyloid deposition, and neurofibrillary lesions and examine the possible influences of ApoE genotype. These relationships will be studied prospectively in transgenic mice expressing mutant presenilin 1 (PS1) and/or mutant amyloid precursor protein (APP), which exhibit neuronal LS abnormalities resembling those in early AD. Combining EM and LM morphometry and cell biological approaches, we will examine the antecedents of the LS abnormalities in the transgenic mice and in FAD fibroblasts expressing mutant PS1 by evaluating the endocytic pathway and characterizing, for the first time in brain, two other routes to lysosomes-autophagy and the direct conversion of endoplasmic reticulum to lysosomes. Disease relevance of these findings will be confirmed in AD tissue. To establish relationships between altered LS function, neurodegeneration, and Abeta production/clearance in vivo, we propose to accentuate Alzheimer pathology in transgenic mice by modulating LS cathepsin inhibitor, cystatin B, in crosses with cystatin B knock-out mice. To address the origins of the lysosomal response, we will identify forms of injury that may stimulate this response in neurons in culture (or endothelial cells) including ischemia, glucose deprivation, hypoxia, oxidative stress, nutrient depletion, and Abeta toxicity. Finally, to define how LS dysfunction promotes or triggers cell death, we will selectively disrupt lysosomal membranes by targeted photo-oxidation to initiate cell death and will characterize the resulting cascade of events leading to death and their relevance to AD. Together, these studies will clarify the importance of the LS in neurodegeneration in AD, generate improved models of AD pathology, and identify novel strategies for therapeutic intervention.